Troubleshooting ELISAs

The enzyme-linked immunosorbent assay (ELISA) is a powerful technique for identifying and quantifying proteins. The assay is less complex than other assays, however problems can still arise when performing ELISAs. These problems can lead to little or no signal, or a high background that obscures the true signal.

This troubleshooting guide lists common issues associated with ELISAs and solutions to these issues.

Poor signal

Low signal can occur for a variety of reasons preventing detection of the target protein.

Not enough target protein in the well

A threshold of detection must be reached before the true signal can be detected above background. Sufficient target protein must be used to achieve this threshold. To increase target protein amounts:

Concentrate samples prior to adsorption to the plate.

Use asandwich ELISAto capture and bind the target protein to the well.

If the target protein/peptide isn’t absorbing well to the plate either:

Conjugate the antigen to a carrier protein before coating the plate

Biotinylate the protein and use streptavidin to adsorb the protein to the plate

Use a positive control.

Titrate the positive control to determine the limit of detection of the assay.

Signal too low

Low signal can arise from the inefficient detection of the target protein. To increase the signal:

Use highly specific antibodies.

Use an indirect detection method to enhance the signal.

Incorrect orientation of the target protein

When using an ELISA to detect antibodies, the antibodies can orient improperly on the plate such that the binding site is no longer accessible to the detecting antibody. To properly orient antibodies, first coat the wells with a protein that binds to the Fc region of the antibody (Protein A or G).

Incubations too short

Short incubations can interfere with formation of antigen-antibody complexes and inhibit color development.

Increase incubation time with antibodies to allow complex formation.

Optimize substrate incubation time.

High background/False Positives

High background results in a low signal-to-noise ratio and an inability to properly detect true signal. Several factors can contribute to a high background.

Matrix effect

Matrix effects occur when the composition of the sample leads to high background or false positives. Certain samples, such as those from plasma and serum, are more prone to matrix effects. To eliminate or test for matrix effects:

Dilute samples using the same diluent as the standard curve.

Use a range of concentrations of the sample.

Cross-reactivity

Cross-reactivity occurs when the antibody recognizes another protein in the sample that has a high homology to the target protein. To avoid cross-reactivity:

Use the most specific antibody available.

Use a more specific ELISA, such as a sandwich ELISA.

Try an antibody that recognizes a different epitope.

Non-specific binding

Non-specific binding refers to binding of the assay antibodies in a manner not related to the specificity of the antibody. Non-specific binding can occur when antibodies bind to the wells or to a component of the blocking or wash buffers. To prevent non-specific binding:

Make sure wells are completely blocked by incubating with an appropriate blocking buffer for the specified time.

Optimize the blocking and washing buffers to make sure they are compatible with the antibodies.

Increase the salt concentration or add detergent to the washing buffers.

Too much/wrong antibody is used

Too much antibody or use of the wrong secondary antibody in an indirect ELISA can lead to overall high background.